Assessment of Radiolabelled Derivatives of R954 for Detection of Bradykinin B1 Receptor in Cancer Cells: Studies on Glioblastoma Xenografts in Mice

Bradykinin B1 receptor (B1R) has garnered attention as a cancer therapeutic and diagnostic target. Several reports on radiolabelled derivatives of B1R antagonists have shown favourable properties as imaging agents in cells highly expressing hB1R following transfection. In the present study, we assessed whether radiolabelled probes can detect B1R endogenously expressed in cancer cells. To this end, we evaluated 111In-labelled derivatives of a B1R antagonist ([111In]In-DOTA-Ahx-R954) using glioblastoma cell lines (U87MG and U251MG) with different B1R expression levels. Cellular uptake studies showed that the specific accumulation of [111In]In-DOTA-Ahx-R954 in U87MG was higher than that in U251MG, which correlated with B1R expression levels. Tissue distribution in U87MG-bearing mice revealed approximately 2-fold higher radioactivity in tumours than in the muscle in the contralateral leg. The specific accumulation of [111In]In-DOTA-Ahx-R954 in the tumour was demonstrated by the reduction in the tumour-to-plasma ratios in nonlabelled R954-treated mice. Moreover, ex vivo autoradiographic images revealed that the intratumoural distribution of [111In]In-DOTA-Ahx-R954 correlated with the localisation of B1R-expressing glioblastoma cells. In conclusion, we demonstrated that [111In]In-DOTA-Ahx-R954 radioactivity correlated with B1R expression in glioblastoma cells, indicating that radiolabelled derivatives of the B1R antagonist could serve as promising tools for elucidating the involvement of B1R in cancer.


Introduction
Kinins are a family of oligopeptides from the kallikrein-kinin system, generated in the inflammatory milieu of the tissue microenvironment, and are involved in numerous pathophysiological processes, including the regulation of blood pressure and inflammatory processes, pain sensation, and cell proliferation and migration [1,2].Kinins signal through the activation of two G-protein-coupled receptors: inducible bradykinin receptor B1 (B1R) and constitutive receptor B2 (B2R).Activated kinin receptors in the cancer microenvironment are thought to be involved in tumour growth, angiogenesis, invasion, and cancer metastasis [3,4].In particular, B1R is considered an important therapeutic target due to its inducible expression.However, it has been reported that the expression patterns and functions of B1R differ depending on the type of cancer.For instance, normal levels of B1R were observed in oesophageal carcinoma when compared with normal tissues, whereas increased levels of B1R characterise colorectal adenomas [5,6].The function of B1R as a suppressor of cancer progression has been observed in melanoma cells [7], whereas its function as a modulator has been reported in colorectal, prostate, and breast cancers [8][9][10].In studies of mice with intrastriatally implanted glioma cells, B1R selective antagonist-treated mice and B1R knockout mice showed a remarkable increase in tumour invasiveness, as indicated by tumour size or mitotic index [11].However, combined treatment with B1R and B2R antagonists and B1R/B2R double knockout mice showed decreased tumour invasiveness, although pharmacological and genetic B2R blocking was not effective.Recently, stem cell-based therapies have gained popularity in the treatment of various diseases, including cancer.There are few reports on the role of B1R in the interactions between glioblastoma cells and mesenchymal stem cells, which are considered a promising source of stem cells for various therapies [12].Thus, B1R in the cancer microenvironment represents an important therapeutic target, as it plays a role in regulating cancer progression.However, the efficacy of B1R regulation in cancer treatment remains unclear.
Imaging techniques capable of detecting B1R may be beneficial for investigating in vivo B1R changes in the cancer microenvironment.Nuclear medicine imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), effectively allow for the noninvasive visualisation and measurement of physiological processes using radioactive molecular probes.Several reports have successfully developed PET probes that leverage B1R antagonist sequences [13][14][15][16][17][18].
[ 68 Ga]Ga-DOTA-dPEG2-R954 ( 68 Ga-HTK01083) and [ 18 F]F-AmBF 3 -Mta-dPEG2-R954 ( 18 F-HTK01146) are PET probes developed by Kuo et al. and are based on the properties of R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-D-2-Nal-Ile), which is associated with favourable pharmacokinetics, stability, and safety profile [17].PET studies with the probes conducted in mice bearing hB1R-expressing HEK293T tumours demonstrated high-contrast images of tumours, thus indicating that R954 is an effective lead sequence for the optimisation of B1R tracers for cancer imaging.Conversely, most of the previous probes targeting B1R have been evaluated using cells that highly expressed B1R following transfection.Aiming for clinical applications, it is important to clarify whether the probes can detect B1R expression levels comparable to those in patients.Thus, assessing this using cancer cell lines that endogenously express B1R could prove valuable.
To evaluate radiolabelled derivatives of B1R antagonists, we focused on the involvement of B1R in glioblastoma cells and considered using two typical glioblastoma cell lines (U87MG and U251MG), which are commonly used as experimental models of glioblastoma and have been reported to express B1R [19][20][21].Glioblastoma is the most aggressive primary tumour of the central nervous system.It poses a significant challenge for effective therapy due to its high intra-and intertumoural heterogeneity, rapid invasion, and the presence of therapy-resistant subpopulations of glioblastoma stem cells and their inherent plasticity [22,23].B1R promotes glioblastoma development by supporting the migration and invasion of glioblastoma cells [21].Additionally, studies using co-culture models with bone marrow-derived mesenchymal stem cells showed an increase in B1R expression in U87MG cells, which was correlated with their enhanced invasiveness [24].Thus, B1R as a potential regulator of glioblastoma has been demonstrated in some experiments, which regarded B1R as a potential biomarker for targeting in cancer therapy.Therefore, imaging techniques capable of detecting B1R are valuable for clinical research and basic glioblastoma research.
In this study, we aimed to evaluate the ability of radiolabelled derivatives of a B1R antagonist to detect B1R in tumours derived from glioblastoma cell lines that endogenously express B1R.We synthesised 111 In-labelled derivative of R954, in which the Nterminal acetyl group of R954 was replaced with aminohexanoic acid (Ahx) as a spacer, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was attached to the Ahx ([ 111 In]In-DOTA-Ahx-R954) (Figure 1).We used [ 111 In]In-DOTA-Ahx-R954 as an example of radiolabelled derivatives of the B1R antagonist based on previous studies that addressed the peptide antagonists of B1R, which are known to tolerate a certain level of N-terminal sequence modifications [25].Furthermore, [ 68 Ga]Ga-DOTA-dPEG2-R954, reported by Kuo et al., previously showed that R954 retains affinity for B1R after the introduction of DOTA to its N-terminus via a spacer [17].To clarify the factors involved in the intratumoural distribution of [ 111 In]In-DOTA-Ahx-R954, we conducted ex vivo autoradiography.We then compared the intratumoural distribution of blood flow as measured using [ 14 C]iodoantipyrine, the expression pattern of B1R, and the presence of glioblastoma cells, as indicated by the glial fibrillary acidic protein (GFAP) marker expressed in astroglial tumours [26,27].We then compared the intratumoural distribution of blood flow as measured using [ 14 C]iodoantipyrine, the expression pattern of B1R, and the presence of glioblastoma cells, as indicated by the glial fibrillary acidic protein (GFAP) marker expressed in astroglial tumours [26,27].

In Vitro Accumulation of [ 111 In]In-DOTA-Ahx-R954 in U87MG and U251MG Cells
Immunoblotting revealed higher B1R expression levels in U87MG cells than in U251MG cells (Figures 2A and S3).In U87MG cells, the accumulated radioactivity of [ 111 In]In-DOTA-Ahx-R954 increased with an increase in the incubation time, reaching 6.29% dose/mg protein after 180 min of incubation (Figure 2B).U251MG cells showed no obvious time-dependent increase in accumulated radioactivity, and the accumulated level was significantly lower than that in U87MG cells at all time points.Nonlabelled R954 reduced the accumulated radioactivity in both cells.The decreased rates relative to the control group were 68.3% and 21.6% for the U87MG and U251MG cell lines, respectively (Figure 2C).In the blocking study with the B1R agonist [des-Arg 10 ]-kallidin, a significant decrease in radioactivity was observed in U87MG cells but not in U251MG cells.Increasing concentrations of [ 111 In]In-DOTA-Ahx-R954 were incubated with U87MG and U251MG cells in the presence or absence of nonlabelled R954 (Figure 3).In U87MG cells, the specific binding curve of [ 111 In]In-DOTA-Ahx-R954 reached saturation, and Scatchard analysis demonstrated that [ 111 In]In-DOTA-Ahx-R954 bound to a single class of sites with a Kd of 54.1 ± 18.9 nM and maximal binding (Bmax) of 0.95 ± 0.79 pmol/mg protein.Since the specific binding to U251MG cells was low and did not reach saturation over the range of concentrations examined, the Kd and Bmax values for U251MG cells could not be determined.

Biodistribution of [ 111 In]In-DOTA-Ahx-R954 in U87MG-Bearing Mice
First, we confirmed the plasma stability of [ 111 In]In-DOTA-Ahx-R954 using C57BL/6JSlc male mice; 79.6 ± 3.1% of the radioactivity remained intact in the plasma 60 min after injection (Figure S4).Subsequently, in vivo radioactivity distributions in U87MG-bearing mice were assessed at several time points after injection (Table 1).At 1 h after injection, the accumulated radioactivity in the tumour engrafted in the right hind leg was 2.3-fold higher than that in the muscles of the nontreated left hind leg.Radioactivity levels in the plasma were higher than those in many tissues, including tumours, at 1 h after injection, and they gradually declined over time.At 4 h after injection, the decrease in radioactivity levels in the plasma (98% reduction compared to 1 h) resulted in decreased radioactivity levels in many tissues, including tumours (48% reduction compared to 1 h).Subsequently, the tumour-to-plasma ratios significantly increased at 4 h, and the highest

In Vitro Accumulation of [ 111 In]
In-DOTA-Ahx-R954 in U87MG and U251MG Cells Immunoblotting revealed higher B1R expression levels in U87MG cells than in U251MG cells (Figures 2A and S3).In U87MG cells, the accumulated radioactivity of [ 111 In]In-DOTA-Ahx-R954 increased with an increase in the incubation time, reaching 6.29% dose/mg protein after 180 min of incubation (Figure 2B).U251MG cells showed no obvious timedependent increase in accumulated radioactivity, and the accumulated level was significantly lower than that in U87MG cells at all time points.Nonlabelled R954 reduced the accumulated radioactivity in both cells.The decreased rates relative to the control group were 68.3% and 21.6% for the U87MG and U251MG cell lines, respectively (Figure 2C).In the blocking study with the B1R agonist [des-Arg 10 ]-kallidin, a significant decrease In radioact"vity'was observed in U87MG cells but not in U251MG cells.Increasing concentrations of [ 111 In]In-DOTA-Ahx-R954 were incubated with U87MG and U251MG cells in the presence or absence of nonlabelled R954 (Figure 3).In U87MG cells, the specific binding curve of [ 111 In]In-DOTA-Ahx-R954 reached saturation, and Scatchard analysis demonstrated that [ 111 In]In-DOTA-Ahx-R954 bound to a single class of sites with a K d of 54.1 ± 18.9 nM and maximal binding (B max ) of 0.95 ± 0.79 pmol/mg protein.Since the specific binding to U251MG cells was low and did not reach saturation over the range of concentrations examined, the K d and B max values for U251MG cells could not be determined.ratio was observed at 24 h.Among the main organs, the kidneys exhibited the highest uptake, and the liver and lungs displayed relatively high radioactivity at 1 h after injection.Moreover, the liver, kidney, and large intestine showed increased radioactivity at 4 h, which was in contrast to the decreased radioactivity in most other organs at this time point.At 24 h, the radioactivity in the kidney remained high at 109.2% dose/g tissue.(A) (B)

Biodistribution of [ 111 In]In-DOTA-Ahx-R954 in U87MG-Bearing Mice
First, we confirmed the plasma stability of [ 111 In]In-DOTA-Ahx-R954 using C57BL/6JSlc male mice; 79.6 ± 3.1% of the radioactivity remained intact in the plasma 60 min after injection (Figure S4).Subsequently, in vivo radioactivity distributions in U87MG-bearing mice were assessed at several time points after injection (Table 1).At 1 h after injection, the accumulated radioactivity in the tumour engrafted in the right hind leg was 2.3-fold higher than that in the muscles of the nontreated left hind leg.Radioactivity levels in the plasma were higher than those in many tissues, including tumours, at 1 h after injection, and they gradually declined over time.At 4 h after injection, the decrease in radioactivity levels in the plasma (98% reduction compared to 1 h) resulted in decreased radioactivity levels in many tissues, including tumours (48% reduction compared to 1 h).Subsequently, the tumour-to-plasma ratios significantly increased at 4 h, and the highest ratio was observed at 24 h.Among the main organs, the kidneys exhibited the highest uptake, and the liver and lungs displayed relatively high radioactivity at 1 h after injection.Moreover, the liver, kidney, and large intestine showed increased radioactivity at 4 h, which was in contrast to the decreased radioactivity in most other organs at this time point.At 24 h, the radioactivity in the kidney remained high at 109.2% dose/g tissue.
To hamper the specific accumulation of [ 111 In]In-DOTA-Ahx-R954 in vivo, an excess of nonlabelled R954 was administered to U87MG-bearing mice (Table 2).Simultaneous administration of nonlabelled R954 significantly decreased the accumulation of [ 111 In]In-DOTA-Ahx-R954 in the kidneys at both 1 h and 4 h after injection (60% and 17% decrease, respectively).However, other tissues, including tumours, showed increased R954-induced radioactivity at 1 h after injection.The highest rate of increase was shown in plasma (374%), and the next highest rates were in the muscle (324%) and stomach (315%) tissues.These results suggest that R954 induced an increase in plasma concentrations of [ 111 In]In-DOTA-Ahx-R954, resulting in increased radioactivity concentrations in all tissues except the kidneys.At 4 h after injection, although plasma concentrations in the R954 treatment group decreased compared to 1 h after administration, they remained higher than those in the control group.In most organs except the kidneys, the radioactivity levels of R954-treated mice were comparable to those of the control group.When comparing the plasma concentration ratios, we observed that the R954-treated group showed significant decreases in tumour-to-plasma ratios (30% and 69% reduction at 1 h and 4 h after injection, respectively), indicating the specific accumulation of [ 111 In]In-DOTA-Ahx-R954 in tumours.4A).In addition, the [ 14 C]iodoantipyrine data revealed that blood flow did not change between the tumour and muscle, indicating that the higher accumulation of [ 111 In]In-DOTA-Ahx-R954 in tumours compared to muscles under normal conditions was not dependent on changes in blood flow (Figure 4B).
[ In]In-DOTA-Ahx-R954 had a significant influence on tissue accumulation.To clarify the effect of blood flow on [ 111 In]In-DOTA-Ahx-R954 accumulation in tumours, we compared the intratumoural distribution of [ 111 In]In-DOTA-Ahx-R954 and [ 14 C]iodoantipyrine, a blood flow tracer, visualised using ex vivo autoradiographic images obtained via the double tracer method.Representative ex vivo autoradiographs of [ 111 In]In-DOTA-Ahx-R954 and [ 14 C]iodoantipyrine on identical slides showed different accumulation distributions in the tumour and muscle tissues (Figure 4A).In addition, the [ 14 C]iodoantipyrine data revealed that blood flow did not change between the tumour and muscle, indicating that the higher accumulation of [ 111 In]In-DOTA-Ahx-R954 in tumours compared to muscles under normal conditions was not dependent on changes in blood flow (Figure 4B).

B1R Expression and Intratumoural Distribution of Glioblastoma Cells
Figure 5A shows representative photomicrographs of the U87MG tumour tissue double immunofluorescence labelled for B1R and GFAP, where [ 111 In]In-DOTA-Ahx-R954 showed high accumulation.In this field of view, B1R-positive cells colocalised with GFAP-positive glioblastoma cells.The macro images of GFAP immunoreactivity showed higher fluorescence intensity in the tumour than in the muscle (Figure 5B).Compared to

Discussion
In the present study, we evaluated whether [ 111 In]In-DOTA-Ahx-R954, a radiolabelled derivative of the B1R antagonist, could detect B1R endogenously expressed in glioblastoma cells.Cellular uptake experiments in two cell types with different B1R expression levels, U87MG and U251MG, suggest that the accumulation rate of [ 111 In]In-DOTA-Ahx-R954 reflects the differences in B1R expression levels.U87MG cells showed a higher expression of B1R and an accumulation of [ 111 In]In-DOTA-Ahx-R954 compared with U251MG cells.The blocking effects of the nonradiolabelled B1R antagonist R954 and agonist [des-Arg 10 ]kallidin on [ 111 In]In-DOTA-Ahx-R954 accumulation were also higher in U87MG than in U251MG cells, indicating that the accumulation of [ 111 In]In-DOTA-Ahx-R954 is specific to B1R in glioblastoma.Scatchard analysis from the saturation experiment revealed a single class of binding sites in U87MG cells with a K d of 54.1 ± 18.9 nM and B max of 0.95 ± 0.79 pmol/mg protein.
Based on the favourable results observed in vitro, we evaluated [ 111 In]In-DOTA-Ahx-R954 under in vivo conditions using U87MG tumour-bearing mice.The engrafted tissue in U87MG tumours demonstrated a two-fold higher accumulation of [ 111 In]In-DOTA-Ahx-R954 compared to the muscle in the non-treated leg.The tumour-to-plasma ratios were increased with decreasing plasma concentration in a time-dependent manner after injection of [ 111 In]In-DOTA-Ahx-R954.The value observed at 4 h was comparable to the tumour-to-blood ratio of [ 68 Ga]HTK01083 and [ 18 F]HTK01146 at 1 h post injection [17].However, in a blocking study conducted in mice, simultaneous injection of R954 increased the accumulation rate in most organs, including tumours, at 1 h after injection.This was mainly driven by an increased plasma concentration of [ 111 In]In-DOTA-Ahx-R954 induced by R954.In a previous report on [ 68 Ga]HTK01083, elevations in radioactivity were observed in organs other than the kidneys and tumours derived from B1R-expressing HEK293T cells established through transfection [17].Based on the increased radioactivity in the blood, the authors suggested that the dosage of the administered blocking agent may have saturated the clearance pathways.In our study, conducted under the same administration conditions, R954 significantly decreased the accumulation rate in the kidneys, thus indicating that the renal route, as the main excretion route of [ 111 In]In-DOTA-Ahx-R954, was saturated.Even after 4 h, most organs, except for the kidneys, in the R954 treatment group showed accumulation levels equivalent to those in the control group, suggesting that increased plasma concentration caused nonspecific accumulation.Thus, to correct for the effect of increased plasma concentration, we re-assessed the blocking effect of R954 using the ratio to plasma concentration.Consequently, the corrected accumulation rate in R954-treated mice decreased in most organs.Furthermore, our results indicate reductions in the tumourto-plasma ratios in R954-treated mice (30% and 69% reduction at 1 h and 4 h after injection, respectively), emphasising the specific accumulation of [ 111 In]In-DOTA-Ahx-R954 under in vivo conditions.In addition, the decrease in the corrected accumulation rate in organs, including the heart, lungs, liver, and kidneys, further supports the specificity of [ 111 In]In-DOTA-Ahx-R954 against B1R.This is consistent with previous reports of B1R expression using quantitative real-time RT-PCR of B1R mRNA in mice [28,29].
Our blocking experiments using R954 in mice revealed that the plasma concentration of [ 111 In]In-DOTA-Ahx-R954 had a significant influence on tissue accumulation.Pharmacokinetics in tumours have been reported to be influenced by tumour blood flow [30].Therefore, to elucidate the effect of blood flow on [ 111 In]In-DOTA-Ahx-R954 accumulation in tumours, we compared the intratumoural distribution of [ 111 In]In-DOTA-Ahx-R954 relative to the blood flow, determined using the [ 14 C]iodoantipyrine marker.Ex vivo autoradiographic images obtained using the double tracer method revealed that the in-tratumoural distribution of [ 111 In]In-DOTA-Ahx-R954 was not dependent on the local blood flow in the tumour.In addition, no difference was found in blood flow between tumours and muscles, further indicating that the increase in tumour accumulation of [ 111 In]In-DOTA-Ahx-R954 was not affected by angiogenesis-induced changes in blood flow.Immunohistochemical studies demonstrated the colocalisation of B1R with GFAP, a marker for glioblastoma, in the area where accumulated radioactivity was observed in the autoradiogram with [ 111 In]In-DOTA-Ahx-R954.These results indicate that radiolabelled R954 can detect B1R endogenously expressed in the U87MG glioblastoma cell line, which is valuable for understanding the role of B1R in cancer.
Our study has several potential limitations.First, the binding affinity of [ 111 In]In-DOTA-Ahx-R954 (K d = 54.1 ± 18.9 nM for U87MG cells) was lower than that of previously reported radioligands for B1R, such as [ 3 H](des-Arg 10 , Leu 9 )-kallidin (0.33 ± 0.07 nM and 1.9 ± 0.5 nM for lung fibroblast cell membrane and B1R+ HEK293T cell membrane, respectively) [13,31].Although we could not evaluate the diagnostic utility of [ 111 In]In-DOTA-Ahx-R954 by comparing it with previously reported probes for SPECT and PET using parameters obtained under the same experimental conditions, improving the affinity is important in the development of [ 111 In]In-DOTA-Ahx-R954 as a diagnostic agent.Additionally, the poor blood-brain barrier penetration of [ 111 In]In-DOTA-Ahx-R954 makes it difficult to use for detecting tumours present in the brain.To further develop SPECT probes targeting B1R, small molecule agonists and antagonists targeting B1R [18,[32][33][34] may be useful as lead compounds for B1R imaging probes due to their improved bloodbrain barrier penetrance.Second, the number of glioblastoma cell lines used (U87MG and U251MG) was limited.Although the two cell lines were used as a common model for glioblastoma, using more types of cells that have different B1R expression levels is necessary to clearly determine the characteristics of [ 111 In]In-DOTA-Ahx-R954.Additionally, as bradykinin receptors in glioma cells have been reported to be positively correlated with the World Health Organization tumour grade [11,35], it is important to clarify the correlation between [ 111 In]In-DOTA-Ahx-R954 accumulation and grade.Moreover, the utilisation of a radiolabelled B1R antagonist for studies using glioma stem cell models and coculture systems with mesenchymal stem cells is required for further advancement of research on stem cell-based therapies for glioblastoma.
In summary, the present study demonstrated that [ 111 In]In-DOTA-Ahx-R954 could detect B1R in tumours derived from endogenous B1R-expressing U87MG glioblastoma cells.Nevertheless, the assessment of [ 111 In]In-DOTA-Ahx-R954 as a SPECT probe should be approached with caution, considering its high accumulation in normal tissues, such as the kidneys, and the unsatisfactory tumour-to-background contrast.However, the finding that the accumulation of [ 111 In]In-DOTA-Ahx-R954 is positively correlated with tumour B1R expression levels suggests potential clinical applications for radiolabelled derivatives of B1R antagonists, which have been actively studied as PET/SPECT probes.Moreover, these probes can be valuable for evaluating models with B1R expression patterns similar to those observed in patients.While our experiments were conducted using glioblastoma cells as an example, B1R-targeting probes have broad applications across various cancer types.The expression patterns and functions of B1R differ depending on the type of cancer [5][6][7][8][9][10].Moreover, developing radiolabelled probes using a B1R antagonist holds potential in elucidating the function of B1R in cancers.

In Vitro Assay Using Glioblastoma Cells
Human glioblastoma cell lines U87MG and U251MG were obtained from the American Type Culture Collection (Manassas, VA, USA) and kept under standard cell culture conditions (5% CO 2 , 37 • C).Cells were cultured in Eagle's minimum essential medium (E-MEM; FUJIFILM Wako Pure Chemical Co.) supplemented with 10% v/v foetal bovine serum (BioWest S.A.S, Nuaillé, France), 1% v/v nonessential amino acids (FUJIFILM Wako Pure Chemical Co.), 1 mM sodium pyruvate (FUJIFILM Wako Pure Chemical Co.), and 1% v/v penicillin-streptomycin (10,000 unit-10 mg/mL; FUJIFILM Wako Pure Chemical Co.).Cells were seeded at a density of 1 × 10 5 cells/well in Poly-D-Lysine 24-well plates (Corning Inc., Corning, NY, USA).After 24 h, the medium was removed, and the cells were washed with 500 µL of 0.01 M PBS (+) (FUJIFILM Wako Pure Chemical Co.) and incubated at 37 • C in 500 µL of Hanks Balanced Salt Solution (+) (HBSS (+); FUJIFILM Wako Pure Chemical Co.) for 10 min.After preincubation, 250 µL of HBSS (+) containing [ 111 In]In-DOTA-Ahx-R954 (3.5 nM) was added to each well, and incubation was continued.At 10, 30, 60, 120, and 180 min, the incubation solution was removed, and the cells were washed twice with 500 µL of ice-cold 0.01 M PBS (+) and solubilised in 500 µL of 0.3 M NaOH.The value at 0 min was determined by an assay performed using the same protocol as that used immediately after removing the incubation solution.Cell lysate radioactivity was measured using an auto-well gamma counter (2470 WIZARD 2 ; PerkinElmer, Inc., Shelton, CT, USA), and the protein content of the lysates was measured using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA).The results are presented as the percentage dose per milligram of protein (% dose/mg protein).
To examine the specific uptake of [ 111 In]In-DOTA-Ahx-R954, cells were incubated with [ 111 In]In-DOTA-Ahx-R954 in the presence of 100 µM B1R antagonist R954 (pIC 50 = 10.0 ± 3.1 nM) or B1R agonist [des-Arg 10 ]-kallidin (pEC 50 = 9.7 nM) [37,38] for 120 min.The affinity of [ 111 In]In-DOTA-Ahx-R954 for B1R in the cells was measured using an R954 saturation assay with increasing concentrations of [ 111 In]In-DOTA-Ahx-R954 (range 0.7-358 nM).The Scatchard plot was used to estimate the K d and B max values.The parameters were presented as means of three independent experiments (one assay performed on type I collagen-coated plates and two assays performed on Poly-D-Lysine plates).

Western Blotting
Cell lysates were prepared from cultured U87MG and U251MG cells by homogenisation in ice-cold RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing a Protease Inhibitor Cocktail (Merck KGaA, Darmstadt, Germany).The protein content of the lysates was determined using the BCA method, and 20 µg of protein per lane was subjected to electrophoresis on 5-20% sodium dodecyl sulphate-polyacrylamide gels and then transferred onto a nitrocellulose membrane.After blocking with TBST buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5 mL/L Tween 20) containing 5% skim milk, the membranes were incubated with a rabbit polyclonal anti-human BDKRB1 primary antibody (Proteintech Group, Inc., Rosemont, IL, USA).After repeated washes, the membranes were incubated with an Amersham™ horseradish peroxidase-conjugated anti-rabbit secondary antibody (Cytiva, Marlborough, MA, USA).Immunoreactive bands were detected using Chemi-Lumi One Ultra (Nacalai Tesque Inc.) and an Omega Lum G luminescent image analyser (Aplegen Inc., Pleasanton, CA, USA).The protein-loading control for immunoblotting was β-actin, which was detected by a mouse monoclonal anti-β-actin-peroxidase antibody (Merck KGaA).

Animal Model
All experimental protocols were approved by the Animal Care and Use Committee of Showa Pharmaceutical University and performed in accordance with the Principles of Laboratory Animal Care.Five-week-old BALBc nu/nu male mice (Japan SLC, Inc., Hamamatsu, Japan) were xenografted by subcutaneous injection of U87MG cells (5 × 10 6 cells per 50 µL of culture medium) into their right hind legs.The mice were subjected to biodistribution studies when the tumour weight reached 0.1−0.5 g.

Biodistribution in Glioblastoma-Bearing Mice
The experimental conditions, including the dosage of [ 111 In]In-DOTA-Ahx-R954, were in line with the literature on 111 In-labelled peptide [39] and modified based on preexperiments.Mice bearing U87MG tumour xenografts were injected via the tail vein with 100 µL of [ 111 In]In-DOTA-Ahx-R954 (30-74 kBq; 0.01-0.02nmol) and were sacrificed and dissected 1, 4, and 24 h after administration.For the blocking experiments, 100 µg of R954 was used, which was the same amount used for 68 Ga-HTK01083 by Kuo et al. [17].R954 was co-injected with [ 111 In]In-DOTA-Ahx-R954, and mice were sacrificed and dissected 1 and 4 h after administration.The tissues of interest were removed and weighed, and radioactivity was determined using an auto-well gamma counter.The results are presented as a percentage of the injected dose per gram (% dose/g).

Ex Vivo Autoradiography
Double tracer autoradiography using [ 111 In]In-DOTA-Ahx-R954 and [ 14 C]iodoantipyrine was performed as previously described [40].Mice were intravenously injected with [ 111 In]In-DOTA-Ahx-R954 (74 kBq), whereas [ 14 C]iodoantipyrine (37 kBq, PerkinElmer, Inc.) was injected 59 min after the [ 111 In]In-DOTA-Ahx-R954 injection.Mice were de-capitated 1 min after the injection of [ 14 C]iodoantipyrine, and the tumour and muscle tissues were quickly removed and frozen on dry ice.Sections (50 µm) were prepared using a cryostat (Leica CM1520, Leica Biosystems, Deer Park, IL, USA) and exposed to an imaging plate (Cytiva) for 20 h to obtain [ 111 In]In-DOTA-Ahx-R954 images.After the decay of 111 In, the same sections were re-exposed to an imaging plate for three days to obtain [ 14 C]iodoantipyrine images.Autoradiograms were obtained using the Phosphor Imaging Digitize system of an Amersham™ Typhoon™ scanner (Cytiva).Regions of interest (ROIs) were created on the images, and the radioactivity concentration in each ROI was expressed as photostimulated luminescence ([PSL minus background]/area [mm 2 ]).

Immunohistochemistry
Consecutive tumour and muscle slices from the same object used in the ex vivo autoradiographic study were used for immunohistochemical analysis.The primary antibodies used in this study were rabbit polyclonal anti-human BDKRB1 (LifeSpan BioSciences, Inc., Seattle, WA, USA) and mouse monoclonal anti-GFAP (Merck KGaA).The sections were fixed with acetone:methanol (1:1) for 20 min at −20 • C.After antigen retrieval using HistoVT One (Nacalai Tesque Inc.), sections were blocked with Blocking One Histo (Nacalai Tesque Inc.) for 10 min at 25 • C.After overnight incubation of the sections with the mixed antibodies at 25 • C, the primary antibodies were visualised using either an Alexa Fluor 488-labelled anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific Inc.) or a Cy3-labelled anti-mouse IgG (H+L) secondary antibody (Jackson ImmunoResearch Labs Inc., West Grove, PA, USA).Fluorescence images were captured using a fluorescent microscope (BX50, Olympus, Tokyo, Japan).To evaluate the distribution of GFAP immunoreactive cells in whole tissue sections, we obtained fluorescence images detected by a 532-nm excitation laser and a 580-nm long-pass detection filter with an Amersham™ Typhoon™ scanner.The fluorescence intensity in the slices was determined as linear arbitrary units (LAUs) corrected for background ([LAU-background]/area [mm 2 ]) using Multi Gauge Analysis Software version 3.11 (Fuji Film Co., Tokyo, Japan).

Statistical Analysis
All values were expressed as the mean ± SD (for each group).According to the Student paired or unpaired t-tests, statistical significance was set at p < 0.05.

Conclusions
In this study, we demonstrated that [ 111 In]In-DOTA-Ahx-R954, a radiolabelled derivative of the B1R antagonist, can be used to detect the endogenous expression of B1R in glioblastoma cells.Our results suggest that radiolabelled probes targeting B1R are potentially valuable tools for the detection of differentially expressed B1R.This versatility is essential for expanding its applicability across various cancer types and allowing for the clinical translation of B1R imaging in the future.

Figure 3 .
Figure 3. Binding of increasing concentrations of [ 111 In]In-DOTA-Ahx-R954 to cultured U87MG and U251MG cells.Binding of [ 111 In]In-DOTA-Ahx-R954 to U87MG (A) and U251MG (B) cells in the absence (total binding) or presence (nonspecific binding; NSB) of an excess (100 µM) of R954.Specific binding was obtained by subtracting NSB from total binding.Data are expressed as the mean ± SD (n = 4).

Figure 3 .
Figure 3. Binding of increasing concentrations of [ 111 In]In-DOTA-Ahx-R954 to cultured U87MG and U251MG cells.Binding of [ 111 In]In-DOTA-Ahx-R954 to U87MG (A) and U251MG (B) cells in the absence (total binding) or presence (nonspecific binding; NSB) of an excess (100 µM) of R954.Specific binding was obtained by subtracting NSB from total binding.Data are expressed as the mean ± SD (n = 4).

2. 3 .
Ex Vivo Autoradiography of [ 111 In]In-DOTA-Ahx-R954 and [ 14 C]iodoantipyrine A blocking study using R954 in mice revealed that the plasma concentration of [ 111 In]In-DOTA-Ahx-R954 had a significant influence on tissue accumulation.To clarify the effect of blood flow on [ 111 In]In-DOTA-Ahx-R954 accumulation in tumours, we compared the intratumoural distribution of [ 111 In]In-DOTA-Ahx-R954 and [ 14 C]iodoantipyrine, a blood flow tracer, visualised using ex vivo autoradiographic images obtained via the double tracer method.Representative ex vivo autoradiographs of [ 111 In]In-DOTA-Ahx-R954 and [ 14 C]iodoantipyrine on identical slides showed different accumulation distributions in the tumour and muscle tissues (Figure

FigureFigure 5 .
Figure 5A shows representative photomicrographs of the U87MG tumour tissue double immunofluorescence labelled for B1R and GFAP, where [ 111 In]In-DOTA-Ahx-R954 showed high accumulation.In this field of view, B1R-positive cells colocalised with GFAPpositive glioblastoma cells.The macro images of GFAP immunoreactivity showed higher fluorescence intensity in the tumour than in the muscle (Figure 5B).Compared to the ex vivo autoradiography of [ 111 In]In-DOTA-Ahx-R954 from the same object shown in Figure 4A, the intratumoural distribution of GFAP-positive cells was consistent with the [ 111 In]In-DOTA-Ahx-R954 accumulated distribution.The tumour-to-muscle ratio of the analysed fluorescence intensity in the images of GFAP-positive cells correlated well with the [ 111 In]In-DOTA-Ahx-R954 accumulation (Figure 5C).Pharmaceuticals 2024, 17, x FOR PEER REVIEW 7 of 14
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 3 of 14 reported by Kuo et al., previously showed that R954 retains affinity for B1R after the introduction of DOTA to its N-terminus via a spacer [17].To clarify the factors involved in the intratumoural distribution of [ 111 In]In-DOTA-Ahx-R954, we conducted ex vivo autoradiography.

Table 2 .
Effects of simultaneous administration of R954 on the tissue uptake of [ 111 In]In-DOTA-Ahx-R954 in U87MG-bearing mice.